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Peer-reviewed

Research Article

Critical role of P-Glycoprotein-9 in ivermectin tolerance in nematodes

Abstract

Helminth infections in grazing ruminants are of major concern for animal welfare and cause substantial economic losses, prompting the widespread use of ivermectin (IVM). The emergence of IVM resistance, driven by complex and poorly understood mechanisms, increasingly compromises treatment efficacy. Drug efflux transporters, particularly P-glycoproteins (PGPs), are suspected to contribute to resistance. Yet, the study of their individual and functional role is hindered by their diversity in nematodes. This study aimed to dissect the role of specific PGPs in mediating IVM resistance. Thus, the Caenorhabditis elegans strain IVR10, selected for IVM resistance and reported to overexpress pgps, was used as a model. We generated different IVR10 strains each lacking one of six key pgps, and assessed changes in IVM tolerance. Remarkably, only the deletion of pgp-9 significantly increased IVM sensitivity. Furthermore, transgenic expression of Haemonchus contortus pgp-9.1 rescued the resistant phenotype, demonstrating a conserved function across species. To explore drug dynamics, we developed a fluorescent IVM analog, which revealed reduced drug accumulation in IVR10, a phenotype reversed by pgp-9 deletion. Altogether, these findings show that nematode PGP-9 modulates IVM tolerance in IVR10 by controlling drug efflux and highlight it as a potential therapeutic target.

Author summary

The intensive use of macrocyclic lactones such as ivermectin to control gastrointestinal parasitic nematodes in livestock has led to the emergence of widespread resistance. Understanding the multifaceted processes underlying resistance remains an urgent challenge. In our study, we focus on a family of ATP-binding cassette transporter proteins, P-Glycoproteins, which can actively pump drugs out of cells. To explore their role, we relied on a model of an ivermectin-resistant nematode, the Caenorhabditis elegans IVR10 strain. We used gene knock-out and knock-down approaches to abolish or reduce protein expression, together with a fluorescent derivative of ivermectin that allowed us to visualize drug distribution in the worm. We found that one member of this family, PGP-9, plays an important role in drug response. We show that PGP-9 is expressed at key physiological barriers, such as the intestine, thereby preventing drug accumulation in the nematode. Our work provides new insights into how resistant worms can tolerate treatment, which may help in developing strategies to slow the spread of drug resistance in the future.

Citation: Blancfuney C, Guchen E, Garcia M, Faccini J, Sutra J-F, Ramon-Portugal F, et al. (2026) Critical role of P-Glycoprotein-9 in ivermectin tolerance in nematodes. PLoS Pathog 22(3): e1013355. https://doi.org/10.1371/journal.ppat.1013355

Editor: Richard J. Martin, Iowa State University, UNITED STATES OF AMERICA

Received: July 8, 2025; Accepted: March 12, 2026; Published: March 23, 2026

Copyright: © 2026 Blancfuney et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Source data are provided at: https://entrepot.recherche.data.gouv.fr/dataset.xhtml?persistentId=doi:10.57745/HP17LZ.

Funding: This work was supported by the French National Research Agency (ANR) (grant number ANR-21-CE35-0004-01 to M.A.). This funding supported the doctoral fellowship of C.B. We thank Agreenium, DESSE, and INRAE for the scholarship they granted for a doctoral mobility at McGill University (to C.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Helminth infections remain one of the leading causes of morbidity in humans [1,2] and animals. In small ruminants, these infections are of major concern for animal welfare and production [3], and can lead to massive economic losses [4].

Treatments essentially rely on chemicals such as ivermectin (IVM), a broad-spectrum anthelmintic (AH) belonging to the macrocyclic lactone (ML) family. IVM has been massively administered since its market release in 1981 [5] in animals and in humans. This intensive use has inevitably led to the emergence of drug resistance, now undermining control of animal gastrointestinal parasites, e.g., Haemonchus contortus [6]. Moreover, the emergence of resistance in Onchocerca volvulus [7], a human filariae helminth treated with IVM, is of growing concern. Considering the lack of development of new pharmaceuticals and the increasing spread of IVM resistance, uncovering its resistance mechanisms stands as an important challenge [8].

IVM resistance is widely recognized as a multifactorial phenomenon involving overlapping and interacting mechanisms that remain to be fully elucidated. In general terms, AH resistance is defined as heritable traits based on genetic mutations that enable organisms to survive drug doses that would be lethal to the parental population. Distinct from resistance, drug tolerance is a reversible, non-heritable reduction in drug susceptibility resulting from physiological adaptations to exposure [9]. However, the conceptual distinction between resistance and tolerance can be subtle, as several underlying mechanisms remain incompletely characterized and may encompass both adaptive and genetic components. Accumulating evidence suggests that detoxification systems plays a central role in the development and persistence of resistance in nematodes [9]. A number of studies demonstrated an up-regulation of genes encoding P-Glycoproteins (PGPs), ATP-Binding Cassette efflux pumps of the plasma membrane, in IVM-resistant parasites such as H. contortus [10–13], Parascaris univalens [14] and Teladorsagia circumincta [15]. Their expected contribution points to IVM efflux and a protective role against drug accumulation in the nematodes [16]. Among the evidence supporting this role are the activity modulation of heterologously expressed parasite PGPs in response to AH stimuli [17–20], and their protective role in various toxicity assays [14,21,22]. Furthermore, mammalian PGP inhibitors have been shown to enhance IVM sensitivity in parasitic isolates [23,24]. These data support the notion that helminth PGPs can interact with and transport IVM. However, given the broad repertoire of pgp genes in parasites, their individual contributions in this context are still not fully understood.

Studying ML resistance in parasites proves to be a challenging task because of their complex life cycle and dependence on host propagation. Thus, the free-living nematode Caenorhabditis elegans stands out as a powerful well-known model. The development of IVM-resistant strains [25], knock-down and knock-out strains [26] and transcriptional studies [27,28] have been pivotal in understanding AH action and resistance, and in screening for genes associated with resistance.

In addition to PGPs, other mechanisms may contribute to IVM resistance, notably those involving amphids, chemosensory organs of the nematode. IVM-resistant C. elegans exhibit a dye-filling defect of amphids [27], while IVM-resistant H. contortus show structural alterations of amphids [29,30], both observations suggesting impaired sensory function. Recent studies have also underscored the importance of NHR-8, a conserved nuclear hormone receptor in C. elegans and H. contortus, supporting a critical regulatory role of NHR-8 on xenobiotic response pathways [27,31]. Interestingly, osm-3 mutants display NHR-8-mediated pgp overexpression and are drug-resistant [32]. This suggests that NHR-8 regulates ivermectin response through the modulation of pgp expression.

Investigations of pgps in IVM resistance remain limited to transcriptional studies and heterologous expression in models, and their contribution is poorly characterized in IVM-resistant strains. To address this gap, we investigate the role of specific PGPs by selectively targeting genes that are strongly upregulated in the IVM-resistant strain IVR10 [25,27], and downregulated in the nhr-8 loss-of-function mutant [31], namely: pgp-1, pgp-3, pgp-6, pgp-9, pgp-11 and pgp-13. We assessed the impact of individual pgp deletions on IVM sensitivity in the IVR10 background. We identified PGP-9 as a key candidate, and further characterized the function of the H. contortus ortholog in transgenic worms. Finally, we developed a fluorescent IVM derivative to correlate drug accumulation with the resistant phenotype. This study provides the first direct in vivo evidence that PGP-9 from both C. elegans and the parasitic nematode H. contortus can mediate IVM resistance, establishing its central role in drug efflux and linking PGP activity to reduced drug accumulation at the target organism level.

Results

Dominant role of pgp-9 in IVM tolerance

To evaluate the contribution of each PGP to IVM tolerance in C. elegans, we assessed the IVM sensitivity in IVR10 single knock-out strains for pgp-1, -3, -6, -9, -11, and -13 using a larval development assay (LDA). These pgps were selected among the fourteen expressed in C. elegans based on their overexpression in the IVR10 strain [27] and putative regulation by NHR-8 [31]. IVR10 and N2B were used as resistant and susceptible controls, respectively. Dose-response curves analysis revealed that only the deletions of pgp-3 and -9 significantly increased IVM sensitivity in IVR10 (Fig 1A and 1B). Notably, pgp-9 deletion resulted in a strong shift to the left of the dose-response curve (Fig 1A), supported by a three-fold reduction in IC50 compared to IVR10 (3.98 ± 1.22 versus 11.22 ± 1.98 nM, p < 0.0001) (Table 1); whereas the effect of pgp-3 was relatively moderate (6.37 ± 2.71 nM, p < 0.001) (Fig 1B, Table 1). This was supported by resistance factors (RFs) of 2.5 and 4.0, respectively, underscoring that pgp-9 deletion had the most substantial, yet partial, effect in reducing IVM-resistance in IVR10. In contrast, the individual knock-out strains for pgp-1, pgp-6, pgp-11, and pgp-13 did not impact the IVM susceptibility of IVR10, as indicated by IC50 values and RFs comparable to those of the resistant parental strain (Table 1).

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Table 1. Susceptibilities to ivermectin (IVM) of wild-type Bristol N2 strain, IVM selected strain (IVR10), and genetically modified IVR10 for which pgp-1, -3, -6, -9, -11, and -13 have been deleted, on larval development assay (LDA). IC50: inhibitory concentration 50%. RF: resistance factor, IC50 ratio relative to N2B.

https://doi.org/10.1371/journal.ppat.1013355.t001

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Fig 1. pgp-9 mediates IVM tolerance.

Effects of loss of pgp-9 and pgp-3 on tolerance of IVR10 to ivermectin (IVM) in a larval development assay (LDA) (A and B respectively) and in a motility assay (MA) (C and D respectively). Effect of loss of pgp-9 on tolerance of N2B to IVM in a LDA (E). Dose-response curves to IVM in LDA and MA are compared to N2B and IVR10 as controls. Values represent the percentage of L1 reaching the young adult stage (LDA) or the percentage of young adults maintaining motility (MA) within the presence of increasing concentrations of IVM. Data are mean ± S.D. from 3-13 independent experiments. IC50s for each strain are presented in Tables 1–3.

https://doi.org/10.1371/journal.ppat.1013355.g001

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Table 2. Susceptibilities to ivermectin (IVM) of wild-type Bristol N2 strain, IVM selected (IVR10) strains, and genetically modified IVR10 for which pgp-1, -3, -6, -9, -11, and -13 have been deleted on motility assay (MA). IC50: inhibitory concentration 50%. RF: resistance factor, IC50 ratio relative to N2B.

https://doi.org/10.1371/journal.ppat.1013355.t002

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Table 3. Susceptibilities to ivermectin (IVM) of wild-type Bristol N2 strain and pgp-9 loss-of-function N2B in larval development assay (LDA). IC50: Inhibitory concentration 50%.

https://doi.org/10.1371/journal.ppat.1013355.t003

Since IVM targets glutamate-gated chloride channels (GluCls) in C. elegans [33] and induces paralysis in nematodes, we conducted a motility assay (MA) to compare worm motion in the different strains. As expected, N2B and IVR10 exhibited markedly different motility responses to IVM with IC50 values of 28.50 ± 7.51 and 61.88 ± 16.83 nM, respectively (p < 0.01), corresponding to a 2.2 RF (Fig 1C and 1D, Table 2). Consistent with the LDA results, pgp-9 deletion led to a pronounced increase in IVM sensitivity, with a two-fold reduction in IC50 compared to IVR10 (29.14 ± 4.02 versus 61.88 ± 16.83 nM, p < 0.05), and a shift to the left of the dose-response curve (Fig 1C, Table 2). Importantly, the RF of IVR10(Δpgp-9) appeared to be equal to 1.0, revealing that pgp-9 deletion completely restored IVM sensitivity. Meanwhile, deletion of pgp-3 in IVR10 only led to a modest shift in IVM sensitivity (41.34 ± 12.59 versus 61.88 ± 16.83 nM, ns) (Fig 1D, Table 2). Finally, we confirmed that deletions of pgp-1, pgp-6, pgp-11, and pgp-13 did not impact IVM sensitivity in IVR10, as their deletion did not alter either the motility phenotype, as reflected by their respective RFs comparable to that of the resistant IVR10 strains (Table 2).

Taken together, the LDAs and MAs suggest that loss of pgp-9, in particular, induces hypersensitivity to IVM in the IVR10 background. However, IVR10(Δpgp-9) is a CRIPSR-Cas9 engineered strain. To confirm the causative role of pgp-9 deletion in the IVM hyper-sensitive phenotype, we restored gene expression by expressing pgp-9 under its native promoter in an extrachromosomal array in IVR10(Δpgp-9). Transcription of the extrachromosomal array was first confirmed by single worm RT-qPCR (S1 Fig), with IVR10 as a control. We next evaluated the impact of pgp-9 rescue in IVR10(Δpgp-9) on IVM sensitivity using LDA (S2 Fig). The rescue strain, IVR10(Δpgp-9)(ppgp-9::pgp-9), developed at substantially higher IVM concentrations compared to the knock-out strain. For example, in the presence of 10 nM IVM, only 10% of IVR10(Δpgp-9) reached adulthood, whereas 63% of IVR10(Δpgp-9)(ppgp-9::pgp-9) animals developed to this stage (p < 0.0001). Furthermore, development of the rescue strain did not differ significantly from IVR10, which showed 48% adult development. These results indicate that deletion of pgp-9 is responsible for the IVM hypersensitive phenotype observed in IVR10(Δpgp-9).

We next extended our analysis to two additional IVM-resistant strains previously and independently generated in our laboratory by step-wise exposure of N2B to increasing IVM concentrations: IVR10–2014 and IVR10–2022. Pgp-9 was silenced in both strains by feeding with RNA interference (RNAi), and IVM sensitivity was assessed using LDA. We first validated pgp-9 silencing by confirming a strong decrease (around 80%) in pgp-9 transcripts using RT-qPCR (S4 Fig). IC50 values and dose-response curves were compared to controls fed with HT115-control (expressing the empty RNAi vector). IVR10–2014 and IVR10–2022 exhibited phenotypes similar to the standard strain IVR10 on HT115-control and therefore reflective of worms resistant to IVM, as supported by their respective IC50 values (S3 Table). Pgp-9 silencing induced a moderate but consistent shift to the left of the dose-response curve of each IVM-resistant strain (S3 Fig). This was supported by a significant decrease, up to 1.8-fold, of the IC50 values compared to the control RNAi (S3 Fig, S3 Table).

Finally, to assess the role of pgp-9 in modulating IVM response in the susceptible strain N2B, we investigated IVM sensitivity in a mutant strain for pgp-9. LDA revealed a significant increase in IVM sensitivity supported by a shift to the left of the dose-response curve (Fig 1E) and by a 1.8-fold reduction in IC50 values for N2B(Δpgp-9) compared to N2B (Table 3) (0.86 ± 0.10 nM versus 1.54 ± 0.07 respectively, p < 0.001).

Together, these results demonstrate that pgp-9 deletion increases both developmental and motility-based IVM susceptibility - indicating that pgp-9 plays a central role in maintaining IVM tolerance across multiple independently selected C. elegans IVR10 strains and in IVM response in the wild-type susceptible strain.

Functional conservation of PGP-9 in H. contortus

We next investigated whether the function of PGP-9 in IVM resistance is conserved across parasitic species, particularly in the gastrointestinal nematode H. contortus. We assessed whether Hco-pgp-9.1 (also referred to as Hco-pgp-9) can compensate for the loss of Cel-pgp-9 and restore IVM tolerance in the IVR10(Δpgp-9) C. elegans strain.

To better understand the potential functional conservation of these transporters, we first assessed the identity between the two transporters. Given the high conservation of their nucleotide-binding domains (NBDs), which are involved in ATP binding, we excluded these regions from both sequences to focus on the transmembrane domains (TMDs), which are critical for ligand binding and specificity. Partial protein sequence alignment revealed a 63.7% identity between Cel-PGP-9 and Hco-PGP-9.1 (S5 Fig), supporting a conserved functional role.

We then expressed Hco-pgp-9.1 in the IVR10 pgp-9 knock-out strain under Cel-pgp-9 promoter. Three independent rescue strains were studied: IVR10(Δpgp-9)-R#1, IVR10(Δpgp-9)-R#2 and IVR10(Δpgp-9)-R#3. Transcription of Hco-pgp-9.1 transgene was confirmed by single worm RT-qPCR (S6 Fig) in the three independent rescue strains, and compared to Cel-pgp-9 mRNA expression in IVR10. No statistical differences were observed, suggesting that the transgene expression in transgenic worms was comparable to the physiological expression of Cel-pgp-9 in their IVR10 background counterpart.

As expected, the LDA demonstrated that worms lacking the Hco-pgp-9.1 transgene, i.e., IVR10(Δpgp-9), showed low tolerance to IVM. Indeed, at 4 nM, only 45–57% of the population reached the adult stage (Fig 2). At 8 nM, worm development was nearly completely suppressed, in line with the previously determined IC50 of 3.98 nM for IVR10(Δpgp-9). In contrast, worms expressing the Hco-pgp-9.1 transgene consistently exhibited a significantly higher tolerance to the drug, as revealed by a significantly increased percentage of development in the presence of IVM at 4, 8 and 10 nM (Fig 2, p values < 0.01). At 4 nM, the percentage of development was of 99% (IVR10(Δpgp-9)-R#1), 107% (IVR10(Δpgp-9)-R#2) and 90% (IVR10(Δpgp-9)-R#3). At 8 nM, 71%, 81% and 73%, of the rescue IVR10(Δpgp-9)-R#1, IVR10(Δpgp-9)-R#2 and IVR10(Δpgp-9)-R#3, respectively, reached adulthood. Finally, at 10 nM, 67% (IVR10(Δpgp-9)-R#1), 70% (IVR10(Δpgp-9)-R#2) and 59% (IVR10(Δpgp-9)-R#3) were adults, similar to IVR10, in which 48% of animals developed to adulthood at this concentration (Fig 2).

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Fig 2. Impact of Hco-pgp-9.1 rescue in IVR10 (Δpgp-9) on IVM tolerance.

Effect of transgene expression of Hco-pgp-9.1 on the tolerance of IVR10(Δpgp-9) to ivermectin (IVM) in a modified larval development assay (LDA). This assay was performed on three independent transgenic strains expressing the Hco-pgp-9.1 transgene: (A) IVR10(Δpgp-9)-R#1, (B) IVR10(Δpgp-9)-R#2, and (C) IVR10(Δpgp-9)-R#3. For each IVM concentration, the number of adult worms negative (i.e., IVR10(Δpgp-9)) and positive (i.e., Hco-pgp-9.1 transgene) for the extrachromosomal array was normalized to the number of adult worms of each population present in the DMSO well (full development, dotted line) and expressed as a percentage. The percentages of transgenic animals reaching the adult stage for all concentrations were then statistically compared with those of the internal control strain, IVR10(Δpgp-9) (negative for transgene) (two-way ANOVA followed by Sidak’s multiple comparisons test). Data are mean ± S.D. from 3 independent experiments. Transgenic strain genotype: IVR10[Cel-pgp-9 -; pCel-pgp-9::Hco-pgp-9.1::SL2::mCh::Cel-unc-54; pPD118.3].

https://doi.org/10.1371/journal.ppat.1013355.g002

Overall, these findings demonstrate that the heterologous expression of Hco-pgp-9.1 is sufficient to restore a phenotype equivalent to the IVM-resistant one in IVR10(Δpgp-9), indicating functional conservation between Hco-PGP-9.1 and Cel-PGP-9 in this study.

PGP-9 function is critical in IVM clearance

To further explore the role of pgp-9 in IVM resistance, we developed a fluorescent analog of IVM, F-IVM. This probe enabled direct visualization and quantification of drug accumulation in C. elegans. The synthesis of F-IVM (Fig 3A) yielded a product with 99.9% purity, mainly consisting of two known isomers (B1a: 95.4%, B1b: 2.8%), with minor impurities (1.8%) and traces of native IVM (0.04%). Mass spectrometry comparison of IVM and F-IVM spectra (Fig 3B) demonstrated that the structure of F-IVM resembles that of native IVM. The primary difference lies in the loss of two hydroxyl groups from the tetrahydrofuran ring, resulting in the formation of a benzofuran moiety. This structural change enables electron delocalization due to the presence of conjugated bonds, thus conferring fluorescence to the molecule. F-IVM was stable for over 18 hours at room temperature (22 °C) in DMSO (10 mM) and maintained stability for more than five weeks at –20 °C, as well as during long-term storage at –80 °C in DMSO.

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Fig 3. Synthesis, structural characterization, and in vivo toxicity assessment of fluorescent ivermectin (F-IVM).

(A) Schematic representation of the chemical synthesis of F-IVM from native IVM. MI (1-N-methyl-imidazole), TFA (anhydrous trifluoroacetic acid), and TEA (triethylamine). (B) Daughter scan mass spectrum of F-IVM showing strong similarity to native IVM, confirming structural conservation. (C) Larval development assay (LDA) in C. elegans wild-type (N2B) and ivermectin-resistant (IVR10) strains. Dose-response curves show the percentage of L1 larvae reaching the young adult stage at increasing concentrations of F-IVM. (D) Motility assay (MA) assessing locomotor activity of young adult worms after 240 min exposure to F-IVM (0.003-150 μM) at 21 °C, using the WMicroTracker system. Data are presented as mean ± S.D. from three independent experiments.

https://doi.org/10.1371/journal.ppat.1013355.g003

In addition to these structural differences, a comparative analysis of physicochemical parameters between IVM and F-IVM (S4 Table) reveals that F-IVM has reduced hydrogen-bonding capacity, a similar molecular weight and polar surface, increased lipophilicity, and reduced solubility. Although they differ, the low solubility, neutrality at physiological pH, and high polar surface of both molecules suggest their uptake and efflux dynamics should be conserved and point toward a predominant role of active transporters, while arguing against any significant passive diffusion across membranes. Furthermore, it is expected that the decrease in hydrogen-bonding capacity of F-IVM compared to IVM will reduce affinity binding to the drug target, i.e., GluCls. This is further supported by drug efficacy assessment by LDA, which showed that F-IVM has significantly reduced toxicity compared to native IVM (Fig 3C). The liquid LDA revealed that the susceptible N2B strain exhibited an IC50 of 2.70 ± 0.70 µM for F-IVM, approximately 1000-fold higher than for IVM. The resistant IVR10 strain demonstrated cross-resistance to F-IVM with an IC50 of 9.97 ± 0.76 µM (RF of 2.58). We next investigated the impact of F-IVM on the motility of both strains (Fig 3D). Interestingly, F-IVM had no impact on worm motility of both susceptible and IVM-resistant C. elegans in the range of investigated concentrations (up to 150 µM) compared to IVM. This fluorescent analog, although structurally modified, retains physiochemical features that make it relevant as a probe to study drug distribution and transport-mediated IVM resistance in C. elegans.

In that context, animals were exposed to the probe to examine PGP-9 function in F-IVM transport comparing IVR10(Δpgp-9) and IVR10, with N2B as a control. All three strains showed consistent accumulation of the fluorescent probe in the pharynx, particularly in the posterior pharyngeal valve (Fig 4A). A closer examination of N2B and IVR10(Δpgp-9) confirmed that the strains displayed a strong signal in the terminal bulbs of the pharynx, as well as the pharyngeal-intestinal valve (Fig 4B), probably leading to diffusion into pharyngeal muscles.

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Fig 4. pgp-9 modulates F-IVM accumulation in IVR10.

(A) Fluorescent-ivermectin (F-IVM) accumulation in N2 Bristol, IVM-resistant IVR10 and IVR10 pgp-9 knock-out, IVR10(Δpgp-9). Adult C. elegans worms were exposed to F-IVM and then examined by confocal microscopy with a x20 air objective to visualize the fluorescent probe accumulation. Only the fluorescence channel is shown. (B) Magnified images of the probe localization around the pharyngeal-intestinal valve in N2B, IVR10 and IVR10(Δpgp-9). (C) Magnified images of the probe localization in the intestine in N2B, IVR10 and IVR10(Δpgp-9). Brightfield images are included to aid anatomical interpretation. (D) Functional transport assay using IVM fluorescent probe, F-IVM, in N2B, IVR10, and IVR10(Δpgp-9). For each strain, accumulation was quantified in approx. 20–30 adults and experiments were repeated 3 times. Fluorescence was normalized with an untreated population of worms. Data are mean ± S.E.M. * p < 0.05 (one-way ANOVA with Turkey’s post-hoc test).

https://doi.org/10.1371/journal.ppat.1013355.g004

In N2B, F-IVM also accumulated in a punctate pattern at the intestinal region, indicating possible accumulation in intestinal cells or intracellular organelles (Fig 4C), while IVR10 exhibited reduced fluorescence, with minimal or no detectable signal in the intestine (Fig 4A). In contrast, deletion of pgp-9 in IVR10 restored F-IVM accumulation in the gut, though the distribution pattern differed from that observed in N2B. In IVR10(Δpgp-9), the fluorescent signal was predominantly aligned along the intestinal tract, likely corresponding to accumulation at the apical membrane of intestinal cells (Fig 4C).

Fluorescence intensity measured for each strain supported these observations (Fig 4D). The IVR10 strain exhibited significantly reduced F-IVM accumulation, with a two-fold decrease compared to N2B (2.59 ± 0.25 versus 5.40 ± 0.60, p < 0.05). To rule out reduced drug uptake as an explanation for this difference, we assessed pharyngeal pumping activity (S7 Fig). N2B and IVR10 displayed similar pumping rates, with mean frequencies of 3.62 and 3.55 Hz, respectively (ns).

Importantly, pgp-9 deletion in IVR10 restored F-IVM accumulation to levels comparable to the N2B susceptible strain (5.72 ± 0.84 versus 5.40 ± 0.60, ns). Taken together, these results show a strong relationship between F-IVM accumulation and the level of IVM resistance in each strain, highlighting PGP-9 as a critical determinant of F-IVM accumulation in the resistant model IVR10.

Strategic tissue localization of PGP-9

To further elucidate the physiological contexts in which pgp-9 deletion leads to increased F-IVM accumulation, we examined the tissue-specific expression pattern of the pgp-9 gene in N2B(Δpgp-9) and IVR10(Δpgp-9) using a pgp-9 promoter-mCherry (mCh) fusion construct. mCh was localized in the pharyngeal bulbs (anterior and posterior), as well as in the intestine throughout all life stages (L1 to adulthood) (Fig 5). Interestingly, mCh expression was also detected at the periphery of the pharynx muscle, in an area where neuron cells, and notably amphids, are typically localized (“N” arrow in Fig 5). Expression patterns were similar in N2B (Fig 5A) and IVR10 (Fig 5B), underlining a conserved tissue-specific localization between these two strains. In summary, our data suggest that PGP-9 is mainly expressed in the pharynx, in the gut, and neurons, independently of the resistance status.

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Fig 5. mCh driven expression by pgp-9 promoter.

Expression patterns were observed in N2B(Δpgp-9) (A) and IVR10(Δpgp-9) (B). Strains were observed using confocal fluorescence microscopy. Expression was observed in all life stages, mainly in the pharynx anterior and posterior bulbs and in the intestine, as shown by the arrows. Strains also demonstrated expression of mCh in the area where neuron cells are localized. Arrows indicate: P: pharynx; I: intestine; N: neurons. Transgenic strain genotype: N2B[Cel-pgp-9 -; pCel-pgp-9::SL2::mCh::Cel-unc-54; pSC391]; IVR10[Cel-pgp-9 -; pCel-pgp-9::SL2::mCh::Cel-unc-54; pPD118.3].

https://doi.org/10.1371/journal.ppat.1013355.g005

Discussion

Understanding the functional basis of IVM resistance in nematodes remains a major challenge. In this study, we provide new insights into the role of nematode PGPs in this context. We relied on the C. elegans IVR10 strain, generated by IVM stepwise selection pressure, approaching real-life patterns of drug exposure and offering a therapeutically relevant resistant model. Our strategy involved targeted deletions of pgp genes in C. elegans directly within the IVM-resistant background using CRISPR-Cas9. Thereby, we generated IVR10-derived strains with full-length individual deletions of pgp-1, pgp-3, pgp-6, pgp-9, pgp-11, and pgp-13. These genes were previously reported to be upregulated in IVR10 [27] and downregulated in the nhr-8 mutant [31], making them prime targets of interest in the context of IVM resistance. This approach allowed us to directly assess the contribution of each transporter to the IVM-resistant phenotype. Our study identified pgp-9 as a key player in IVM tolerance and provided functional evidence of its involvement in modulating IVM efficacy in drug-resistant C. elegans. Indeed, the genetic deletion of pgp-9 in the IVR10 resistant background significantly increased IVM efficacy. This role was further validated by pgp-9 rescue in the hypersensitive strain IVR10(Δpgp-9), and by RNAi-mediated knock-down in two other independently derived resistant strains, IVR10–2014 and IVR10–2022.

Importantly, the functional importance of PGP-9 in mediating IVM tolerance was demonstrated using two distinct phenotypic assays: LDA and MA. Interestingly, the extent of restored IVM sensitivity differed between the tests. In the LDA, deletion of pgp-9 substantially increased IVM sensitivity but did not fully return to N2B levels while it did in the MA. This aligns with previous observations [34], suggesting that pgp-9 may play a specific non-redundant role in mediating particular physiological responses to IVM, notably those affecting nematode motility. The discrepancy between these assays underscores the complexity of resistance phenotypes and the importance of using complementary approaches when evaluating gene function in drug resistance.

In contrast, deletions of the other pgps had negligeable effects on IVM tolerance, indicating redundant and limited roles in our resistant background. Intriguingly, although pgp-6 knockdown was previously shown to modulate IVM sensitivity in IVR10 C. elegans [31], its deletion had no significant effect in our study, possibly reflecting a context-dependent role.

The contribution of pgp-9 in IVM tolerance was further supported using a loss-of-function N2B strain, as previously described [26], and through RNAi-mediated knock-down in two additional IVM resistant strains. The effect of RNAi was more moderate compared to the knock-out, likely due to incomplete gene silencing [35]. Overall, these results are partially consistent with previous findings in N2B strains [26]. Janssen et al. [26] study suggests that all pgps, with differential extents, contribute to basal IVM detoxification in the wild-type strain, in particular: pgp-1, -3, -9, -11, -12, -14. However, in IVR10, only pgp-9 and pgp-3 were involved in IVM tolerance mechanisms. This likely reveals differences related to the susceptible and resistant genetic context: in N2B, where no resistance gene has been selected, multiple pgps may be mobilized in response to IVM, and deletion of any single transporter may compromise the detoxification capacity. By contrast, in IVR10 worms, in which resistant alleles have been selected and spread within the population, tolerance mechanisms may rely on a narrower set of dominant pgps, with pgp-9 playing an important role, which couldn’t have been revealed in a wild-type strain. In addition, the pleiotropic function of some pgps may complicate results interpretation. For instance, pgp-14 has been implicated in lipid transport during pharynx morphogenesis [36], and its deletion was reported to cause developmental delays independently of IVM exposure [26]. Future work should extend these analyses to other resistant strains and additional drugs, using RNAi knock-downs to assess whether the same set of pgps dominates tolerance across resistant backgrounds. In parallel, investigating ubiquitous or tissue-specific over-expression of pgp-9 would help better define its contribution to IVM response in resistant-IVR10 versus susceptible-N2B.

Our phylogenetic tree (S8 Fig) highlights clusters of closely related PGPs, suggesting functional redundancy. Our results point to a more complex scenario. For instance, although PGP-1 is the closest homolog of PGP-9 in C. elegans, its deletion did not affect IVM sensitivity in IVR10. Despite their phylogenetic proximity and colocalization in the intestine, pgp-1 and pgp-9 still exhibit distinct cellular expression patterns: pgp-9 is additionally expressed in neurons and coelomocytes [37]. Furthermore, they may also differ in terms of expression across developmental stages or protein level, trafficking, or ATPase activity. Our observations indicate that PGP-9 fulfills specific physiological functions, including IVM transport. Consistently, Zhao et al. previously characterized individual Cel-pgp localizations, supporting tissue-specific expression and function for each pgp [38]. Even though the functional redundancy among PGPs remains poorly understood to date, some may indeed play a redundant role, provided that their functional similarity and key features cited overhead are conserved. A limitation of our study is that it focuses on individual deletions, which could mask the contribution of some pgps through compensatory mechanisms. Thus, combinatorial deletions (e.g., pgp-9 with pgp-3 or pgp-6) should be considered to better assess potential additive or compensatory effects within the pgp family.

To assess the evolutionary conservation of PGP-9 function in a parasitic species, we then heterologously expressed Hco-pgp-9.1 in IVR10(Δpgp-9) worms. This restored the IVM-resistant phenotype, unequivocally demonstrating that H. contortus PGP-9.1 can substitute for C. elegans PGP-9 thus conferring IVM tolerance. A previous study conducted in mammalian cells demonstrated the ability of heterologous Hco-PGP-9.1 to transport Rhodamine 123, a well-known PGP substrate, and its capacity to interact with MLs [18]. Similarly, a yeast-based growth assay indicated that Pun-PGP-9 confers protection against ketoconazole toxicity [14], a fungicide also known to interact with PGPs. However, these studies did not inform on the specific contribution of PGP-9 to xenobiotic clearance in a drug-resistant strain. Our study builds upon this foundation by providing the first in vivo functional demonstration of a conserved detoxification role for a parasitic nematode PGP in a ML-resistant genetic background. While this supports the functional conservation of PGP-9 across nematodes, extrapolating findings from C. elegans to parasitic nematodes such as H. contortus requires caution. Although our phylogenetic tree (S8 Fig) illustrates the strong conservation of the PGP family across these two nematode species and identifies Hco-PGP-9.1 as the closest ortholog of Cel-PGP-9, comparative localization studies will be essential to determine whether tissue- or species-specific expression patterns of PGPs could lead to distinct physiological or phenotypic outcomes.

Evidences suggest IVM resistance in nematodes is partly driven by detoxification systems including PGPs, but IVM dynamics in vivo still remain poorly understood due to a lack of tools to track the drug at tissue level. To that effect, we developed F-IVM, a fluorescent IVM analog. Although the structural differences between the two compounds reduced drug potency, F-IVM exhibits physicochemical properties indicating a conserved behavior in uptake and efflux dynamics compared to the parental molecule, IVM, which provides a rationale for its use to study IVM-tolerance mediated by PGPs in C. elegans. In N2B animals, F-IVM strongly accumulated in the pharynx and intestine, major sites for xenobiotic uptake and metabolism [39]. In contrast, resistant IVR10 worms showed minimal fluorescent drug accumulation. This differential accumulation could not be explained by a potential reduced drug uptake by IVR10, as evidenced by the equivalent pharyngeal pumping activity of N2B and IVR10. Instead, these results suggest an enhanced F-IVM efflux or metabolism in the IVM-resistant strain. This is further supported by a transcriptional study showing the upregulation of several detoxification genes in IVR10 [25,27], thereby suggesting activation of an IVM detoxification pathway. Strikingly, pgp-9 deletion restored F-IVM accumulation, particularly at the pharyngeal-intestinal valve and along the intestinal lumen, pointing to disrupted efflux and possible retention at the apical membrane of intestinal epithelial cells in the absence of PGP-9. Quantitative analysis confirmed increased F-IVM uptake in both IVR10(Δpgp-9) and N2B compared to IVR10. However, accumulation patterns in N2B and IVR10(Δpgp-9) did not fully overlap, indicating additional detoxification components are likely involved in IVM clearance [9]. These findings reinforce the idea that PGP-9 acts as a gatekeeper, limiting IVM bioavailability at pharmacologically active tissues.

We then investigated pgp-9 localization to gain insight on its function in drug clearance. Pgp-9 localization was consistent with previous studies in C. elegans (S5 Table) [37,38,40–42], showing expression in the pharynx and intestine, major sites of drug uptake and detoxification. In H. contortus, distinct tissue distribution of one PGP-9 paralog (e.g., uterine expression) suggests paralog-specific physiological roles [18]. Our results are also consistent with another study showing that tissue-specific overexpression of Pun-PGP-9 in C. elegans confers resistance to MLs in a context-dependent manner [21]. Intestinal expression protected primarily against ingested IVM, while epidermal expression affected drug penetration independently of ingestion. Our study also showed significant pgp-9 expression in C. elegans head neurons, which aligns with scRNA-seq data (S5 Table) [37,40,41] and supports the notion of a conserved neuronal detoxification function across nematodes, notably in P. univalens [14]. Interestingly, it was previously shown that C. elegans and H. contortus with dye-filling defects of amphids exhibited drug resistance, and that in a dye-defective osm-3 mutant, pgp overexpression regulated by NHR-8 conferred protecting against tunicamycin. Therefore, PGP-9 may contribute to protection against toxic xenobiotics, including IVM, in head neurons which are widely enriched in GluCls, the primary IVM target [43]. These findings support a model in which PGP-9 reduces drug entry at physiological barriers, with localization likely influencing its substrate specificity and impact on resistance. Overall, these data support that PGP-9 is located on strategic physiological barriers, and therefore influences IVM accumulation and susceptibility.

Conclusion

Altogether, our study provides the first in vivo functional validation of pgp-9 in IVM resistance in C. elegans and supports a conserved function of PGP-9 between C. elegans and H. contortus. We established that pgp-9 contributes to maintaining IVM resistance by limiting IVM accumulation at uptake sites, and thus acting as a detoxifier at strategic physiological barriers. Our results underline an important role of PGP-9 in regulating IVM concentration in tissues where its effects on GluCl receptors induce toxicity to the nematodes. These findings identify PGP-9 as a potential therapeutic target in efforts to counteract IVM resistance, marking a step forward in our understanding of resistance mechanisms in the C. elegans IVR10 strain. Nonetheless, the multifactorial nature of resistance, which likely implicates other PGPs, CYP450s [44], and regulators like NHR-8 [31,32] and CKY-1 [45,46] warrants further investigation. In particular, examining the role of pgps across the other IVR10-derived strains will be essential to determine whether their contribution to IVM tolerance is limited to the ones identified in this study. Future work should investigate how these networks interact, whether they operate concomitantly or independently, and determine at which stages of tolerance and resistance development they become critical. Exploring combinatorial inhibition of these effectors to target parasitic nematodes could offer promising therapeutic strategies.

Methods

Materials

All chemicals were obtained from Sigma-Aldrich, unless otherwise stated. IVM and its fluorescent analog, F-IVM, were dissolved in DMSO, and the maximal concentration of DMSO was 0.5% in all assays (except for F-IVM, which was of 1% in the liquid LDA).

Caenorhabditis elegans strains and cultivation conditions

Wild-type C. elegans strain N2 Bristol (N2B) and the OP50 Escherichia coli strains were provided by the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minnesota, Minneapolis, MN, USA). Mutant strain for pgp-9 (tm0830), referred as N2B(Δpgp-9), was obtained from the National BioResource Project (Tokyo, Japan). The IVM-resistant strain IVR10 was kindly provided by C. E. James [25].

IVR10–2014 and IVR10–2022 are two IVM-resistant strains that were also selected by step-wise exposure of N2B to increasing concentrations of IVM [31]. IVR10 strains deleted individually for a pgp gene, i.e., IVR10; pgp-1 (knu1129 [Δ7392 bp]), IVR10; pgp-3 (knu1141 [Δ5404 bp]), IVR10; pgp-6 (knu1125 [Δ5208 bp]), IVR10; pgp-9 (knu1140 [Δ9179 bp]), IVR10; pgp-11 (knu1142 [Δ7825 bp]) and IVR10; pgp-13 ((knu1145 [Δ5143 bp]) were designed by InVivo Biosystems (Eugene, Oregon, USA). Full-length deletions were done in the IVR10 genome using CRISPR-sdm transgenesis method, and a three-frame stop sequence was inserted. In this study, these knock-out strains are referred as IVR10(Δpgp-1), IVR10(Δpgp-3), IVR10(Δpgp-6), IVR10(Δpgp-9), IVR10(Δpgp-11) and IVR10(Δpgp-13). The genotype of each strain was confirmed by PCR and sequencing.

Transgenic strains were generated through microinjection via standard protocols as described in the “Generation of transgenic C. elegans” section.

All strains were cultured and handled according to the procedures previously described [27,31]. Nematodes were cultured at 21 °C on Nematode Growth Medium (NGM) agar plates (1.7% bacto agar, 0.2% bacto peptone, 50 mM NaCl, 5 mg/L Cholesterol, 1 mM CaCl2, 1 mM MgSO4, and 25 mM KPO4 Buffer) seeded with E. coli strain OP50 as a food source. All C. elegans strains were cultured on classic NGM agar plates, except for IVR10, which was cultured on NGM plates containing 10 ng/ml of IVM.

Nematodes were synchronized through egg preparation with sodium hypochlorite. An asynchronous population was collected from NGM plates with M9 buffer (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 0.25 g MgSO4 7H2O in 1 l of water). All larval stages except eggs were lysed with bleaching mix (5 M NaOH, 1% sodium hypochlorite) followed by M9 washes. C. elegans eggs were then hatched overnight with mild agitation at 21 °C in M9 to obtain a synchronized population of first-stage larvae (L1).

Larval development assays (LDAs)

This assay measures the potency of IVM to inhibit the development of C. elegans from L1 to young adult. The LDA was essentially conducted as described previously [27,31].

Solid LDA.

Briefly, 30 synchronized L1 larvae were added per well of a 12-well plate. These were seeded on NGM containing increasing concentrations of IVM seeded with OP50 bacteria. Each concentration was set-up in triplicate. Plates were incubated at 21 °C until L1 of the negative control had developed into young adult worms. Development was calculated as a percentage of young adults in the presence of IVM normalized to the untreated control. All experiments were reproduced in at least three biological replicates. Curve fitting for the LDA (sigmoidal dose-response curve with variable slope) was performed with the GraphPad Prism 8.4.2 software. IC50 values, the concentrations at which 50% of the animals fail to reach the young adult stage, were determined. The resistant factor (RF) was calculated as the IC50 of each strain divided by the IC50 of the wild-type strain.

Liquid LDA.

This assay was performed in liquid media when assessing the ability of an IVM fluorescent analog, F-IVM, to inhibit larval development. In that case, 25 synchronized L1s were seeded in 200 µl of complete liquid S-Basal media seeded with OP50 (5mg/ml). Complete medium was prepared as follows: 50 ml of S-Basal (5.85 g NaCl, 1 g K2HPO4, 6 g KH2PO4 in 1 l of water), 500 µl of Potassium Citrate 1 M pH6 (20 g C₆H₈O₇, H₂O, 293.5 g K₃C₆H₅O₇, H₂O in 1 l of water), 500 µl of Trace Metal Solution (1.86 g C₁₀H₁₄N₂Na₂O₈, 0.69 g FeSO4, 7H2O, 0.2 g MnCl2, 4H2O, 0.29 g ZnSO4, 7H2O, 0.025g CuSO4, 5H2O), 150 µl CaCl2 1 mM, 150 µl MgSO4 1 mM, 50 µl Cholesterol 5 mg/ml. Drug treatment was administered by adding 1–2 µl of F-IVM at increasing concentrations. Plates were incubated at 21 °C under gentle agitation until L1 of the DMSO control had developed into young adult worms. Development, dose-response curves, IC50s and RFs were determined as described above.

RNA interference on pgp-9 in IVR10 strains.

RNA interference (RNAi) was conducted by feeding HT115 bacteria transformed with L4440 vector that produces double-stranded RNA against a targeted gene to the strains, as previously described [31]. HT115 bacteria clones expressing pgp-9 RNAi or the empty vector as control from the Ahringer RNAi library were grown for 8h at 37 °C in LB medium containing ampicillin (50 μg/ml). They were then seeded on NGM plates supplemented with carbenicilin (25 μg/ml) and IPTG (1 mM) to induce RNAi expression. Finally, solid LDA was conducted as described above. The extent of knockdown of pgp-9 mRNA was determined by RT-qPCR.

LDA for transgenic strains.

This assay was adapted from another study [36] and from the solid LDA previously described to characterize the rescue function of Hco-pgp-9.1 and Cel-pgp-9 in the IVR10 pgp-9 knock-out strain. Synchronized L1 larvae were obtained from bleaching a population heterogeneously carrying the Hco-pgp-9.1 or Cel-pgp-9 extrachromosomal array. 60 L1s were then added per well of a 6-well plate seeded with NGM and OP50, and containing increasing concentrations of IVM, from 0 (i.e., DMSO) to 10 nM. Each concentration was set up in sextuplicate for the Hco-PGP-9.1 rescue strain and in triplicate forthe Cel-PGP-9 rescue strain. Plates were incubated at 21 °C until L1 of the DMSO control had developed into young adult worms. The number of worms negative and positive for the extrachromosomal array, respectively IVR10(Δpgp-9) and the Hco-PGP-9.1 or Cel-PGP-9 rescue strain, were then counted in each well. The percentage of animals reaching the adult stage for each strain was expressed as a fraction normalized to the number of adult worms of each population present in the DMSO well. All experiments were reproduced in biological triplicate, and three independent transgenic strains of the Hco-PGP-9.1 rescue were studied; IVR10 was subjected to this LDA as a control in duplicate.

Motility assay (MAs)

This assay measures the potency of IVM to inhibit the motility of C. elegans young adults. The MA was essentially conducted as described previously, using the WMicroTracker One device (PhylumTech, Santa Fe, Argentina) [34]. Synchronized young adults (40 per well) were seeded into 200 µl of M9 in a 96-flat well plate. Before each measurement, plates were incubated 15 min at 21 °C to allow the worms to settle. Basal activity (BA) was then measured for 30 min to normalize movement activity in each well. Drug treatment was administered by adding 1µl of IVM at increasing concentrations. Following drug treatment, score activity (SA) was recorded for a 120 min period. Negative controls (NG), i.e., wells without worms were conducted. Motility percentages were calculated for each treated well as fold induction relative to DMSO treated worms which was set to 100, i.e., SA-NC120min/BA-NC30min. All experiments were reproduced in technical triplicates and at least three biological replicates. Curve fitting for the MA (sigmoidal dose-response curve with variable slope), IC50 values, and RFs were determined as explained above.

Electropharyngeogram (EPGs) recordings and pharyngeal pumping activity measurement

EPG recordings for pharyngeal pumping activity measurements were conducted as described elsewhere [31]. Briefly, synchronized worms (N2B and IVR10) were grown until the early adulthood stage. Individuals were collected from their NGM plates and washed with M9 to remove residual OP50 bacteria. They were incubated for 20 min in M9 containing 10 mM serotonin (5-hydroxytryptamine; 5HT). EPGs of individual worms were recorded with the ScreenChip system size 40 (InVivo Biosystems) and the NemAcquire software (InVivo Biosystems) for ~1 min. The experiment was replicated three times, and at least 10 worms were recorded per strain per replicate. EPG recordings were analyzed on the NemAnalysis software (InVivo Biosystems) in order to obtain the mean pharyngeal pumping frequency (Hertz, Hz) of each strain.

RT-qPCR.

A population of 1500 synchronized L1 larvae were added to NGM plates seeded with HT115 or HT115-pgp-9 bacteria in order to assess RNAi efficiency. Non-gravid young adults were then collected using M9 buffer and flashed-frozen in liquid nitrogen in RLT buffer (Qiagen) supplemented with DTT 2 M. Lysed worms were stored -80 °C. Frozen samples were thawed and homogenized three times for 10 sec at 6 m.s−1 in a FastPrep-24 instrument (MP-Biomedicals, NY, USA). Total RNA was extracted using RNeasy Plus Kit (Qiagen, S.A., Courtaboeuf, France) according to the manufacturer’s instructions. Total RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). cDNA was synthesized from 1 μg of total RNA using Maxima H Minus First Strand cDNA Synthesis Kit (Thermofisher).

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using SYBR Green PCR Master Mix (Applied BiosystemsLife Technologies, Courtaboeuf, France) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Gene specific primers that were used are listed in S1 Table. Results are expressed according to the relative quantification method with tba-1 as the reference gene.

Identity between Cel-PGP-9 and Hco-PGP-9.1 and phylogenetic analyses

Predicted amino acid sequences of Cel-PGP-9 (Accession Number: CE15714) and Hco-PGP-9.1 (Accession Number: HCON_00130050–00001) were obtained from WormBase and WormBase ParaSite, respectively. Alignment was first performed with full-length sequences, with the Clustal Omega Multiple Sequence Alignment (MSA) tool (https://www.ebi.ac.uk/jdispatcher/msa/clustalo?stype=protein). Percent Identity Matrices were extracted following the alignment. Nucleotide Binding Domains (NBDs) were then identified with the help of the Scan Prosite tool (https://prosite.expasy.org/scanprosite/) and manually curated from the sequences such as: Cel-PGP9 NBD1(383–619); NBD2(1030–1294) and Hco-PGP-9.1 NBD1(373–609); NBD2(1026–1270). Sequences were then subjected again to Clustal Omega and new identity matrices were obtained.

PGP protein sequences of C. elegans were obtained from WormBase, and of H. contortus from WormBase ParaSite (PRJEB506 [47]). A multiple sequence alignment was performed using Seaview software and the Clustal Omega method (S1 File). A PhyML tree was then generated with the LG model, using aLRT (approximate Likelihood Ratio Test) for branch support, and Hsa-ABCB1 and Mmu-Pgp as an outgroup for tree rooting. Annotations were performed using iTOL (https://itol.embl.de/).

Generation of transgenic C. elegans

Cloning.

Unless stated otherwise, all PCRs were performed with the proofreading Phusion High-Fidelity DNA Polymerase (New England Bioloabs, Ipswich, MA, USA) and all cloning experiments were carried out with the Pro Ligation-Free Cloning Kit (abm, Richmond, BC, Canada), following the manufacturer’s information. Primers used to design the plasmids are listed in S2 Table. A 2.6 kilo base pairs region was amplified from N2B genomic DNA to isolate pCel-pgp-9 and was subcloned into the pMini T2.0 vector (New England BioLabs). This promoter region was then cloned into a PstI/SmaI digested plasmid containing fused SL2::mCherry::Cel-unc-54.

Hco-pgp-9.1 originated from the subcloning vector pGEM-T Easy from Pr. Roger Prichard team (McGill University, Parasitology Institute) [18]. It was cloned into the plasmid containing pCel-pgp-9::SL2::mCherry::Cel-unc-54, between pCel-pgp9 and SL2 (restriction sites SmaI/KpnI) (GeneCust). Plasmid constructs were systematically verified by sequencing (Eurofins).

To rescue Cel-pgp-9 expression in the IVR10(Δpgp-9), a genomic DNA region including the 2.6 kilo base pairs promoter, the pgp-9 and a 3’UTR region were amplified using GoTaq Long PCR polymerase (Promega).

Microinjections.

Plasmid constructs or DNA, i.e., pCel-pgp-9::SL2::mCh::Cel-unc-54, pCel-pgp-9::Hco-pgp-9.1::SL2::mCh::Cel-unc-54 or pCel-pgp-9::Cel-pgp-9::3’UTR, along with co-injection plasmid: pSC391, expressing YFP in neurons, (a gift from Dr. David Miller, Vanderbilt University) or pPD118.3, expressing GFP in the pharynx, (a gift from SEGiCel, SFR Santé Lyon Est CNRS UAR 3453, Lyon, France), were microinjected into either N2B(Δpgp-9) or IVR10(Δpgp-9) gonads (Institute of Parasitology, McGill University, Canada or SEGiCel, SFR Santé Lyon Est CNRS UAR 3453, Lyon, France).

Worms were prepared as described elsewhere [48]. Young adult hermaphrodites N2B(Δpgp-9) were transformed by microinjection of pCel-pgp-9::SL2::mCh::Cel-unc-54 at 75 ng/μL and pSC391 at 15 ng/µl (3:1 ratio).

IVR10(Δpgp-9) were transformed with either pCel-pgp-9::SL2::mCh::Cel-unc-54, pCel-pgp-9::Hco-pgp-9.1::SL2::mCh::Cel-unc-54 or pCel-pgp-9::Cel-pgp-9::3’UTR at 50 ng/µl and pPD118.3 at 5 ng/µl. DNA concentrations were adjusted up to 120 ng/µl using a 1kb Plus DNA ladder (Invitrogen).

Successful transformations were identified using an SMZ800N fluorescent stereomicroscope (Nikon). At least three individual strain lines carrying extrachromosomal arrays were obtained for each construct.

Transgenic strains were maintained by picking regularly GFP or mCh-fluorescent individuals to a new plate, as described in the “Caenorhabditis elegans strains and cultivation conditions” section. Development assays were conducted on the IVR10(Δpgp-9) carrying Hco-pgp-9.1 or Cel-pgp-9 in an extrachromosomal array. Considering Hco-pgp-9.1, three independent strains were studied and are referred to as: IVR10(Δpgp-9)-R#1, IVR10(Δpgp-9)-R#2, and IVR10(Δpgp-9)-R#3. Transcription of Hco-pgp-9.1 and Cel-pgp-9 was confirmed by single worm RT-qPCR. RNA extraction was conducted as described elsewhere [49] and qPCR was performed as explained above.

Fluorescent IVM (F-IVM) probe

Synthesis of F-IVM.

IVM (6 mg, equivalent to 150 µL of IVM diluted to 40 mg/mL in methanol (MeOH)) was mixed with 100 µL of 1-N-methyl-imidazole (MI), followed by sequential additions of 150 µL of anhydrous trifluoroacetic acid (TFA) and 50 µL of triethylamine (TEA), with vortexing after each addition. The reaction mixture was incubated at 70°C for 1 h. The resulting F-IVM was purified using solid phase extraction (SPE) on LC18–100 mg cartridges with an automated SPE robot (Rapidtrace). Cartridges were conditioned with 1 mL MeOH (5 min, 0.2 mL/sec) followed by 1.2 mL H2O (6 min, 0.2 mL/sec). Samples were loaded twice onto cartridges at 0.1 mL/sec for 4.2 min and 4 min, respectively, and left to stand for 1 min. The cartridges were washed sequentially with 2 mL H2O (0.1 mL/sec) and 1 mL MeOH/H2O (25/75) at 0.2 mL/sec, then dried with an air stream (10 min, 0.5 mL/sec). F-IVM was eluted with acetonitrile (AcN, 0.5 mL/sec) and dried under a nitrogen stream (N2) at 60°C. A portion of the eluates was analyzed using an HPLC-Fluorescence system (Ultimate 3000 Thermo, ThermoFisher, Waltham, MA, USA). Samples (25 µL) were injected onto a Supelcosil LC-18 column (150 x 4.6 mm; 3 µm Supelco) under isocratic elution with a mobile phase of H2O (0.4% of acetic acid)/MeOH/AcN (5/30/65, v/v/v) at a flow rate of 1.6 mL/min. The fluorescence detector was set to an excitation wavelength of 355 nm and an emission wavelength of 465 nm. Column temperature was maintained at 40°C, and the autosampler was kept at 20°C.

Characterization of F-IVM.

The structure of F-IVM was confirmed by comparison to native IVM using mass spectrometry (MS). MS analyses were performed in MS scan and daughter scan modes on a triple quadrupole instrument (Xevo TQ, Waters, Milford, MA, USA) with electrospray ionization in positive mode (ESI+). Key parameters included a capillary voltage of 3.5 kV, source temperature of 150 °C, desolvation temperature of 600 °C, and cone voltage of 25 V. Fragmentation was induced using argon as collision gas (collision energy: 20 eV).

F-IVM stability study.

Short-term stability of the F-IVM probe was assessed at room temperature (24 and 48 h) and 4 °C (24 h, 48 h, and 12 days). Long-term stability was evaluated through repeated freeze–thaw cycles and continuous storage at –80 °C for up to one year. Quantification was performed by HPLC-fluorescence as previously described, using a fluorescent ivermectin derivative (IVM-TA) as an external standard. Stability was determined by comparing F-IVM/IVM-TA area ratios to initial values (T₀).

Confocal microscopy

Transgenic strain imaging.

An asynchronous population of transgenic worms was washed once with M9 buffer to remove OP50 bacteria. Worms were paralyzed with 2.5 mM Sodium Azide and mounted on glass. mCherry, expressed under the native promoter of Cel-pgp-9 in N2B and IVR10 pgp-9 knock-outs, was visualized with Zeiss LSM 710 AxioObserver Plan-Apochromat 20x/0.8 M27 or 40x/1.3 Oil Iris (Infinity-INSERM UMR1291, France). mCh was excited at 561 nm and recorded between 581 and 664 nm, and brightfield images were recorded to aid anatomical interpretation.

F-IVM quantification.

Accumulation of the fluorescent F-IVM was investigated in adult N2B, IVR10, and IVR10(Δpgp-9) C. elegans that were grown in liquid media. Synchronized L1s were incubated in complete liquid media S-Basal seeded with OP50 at 21 °C under gentle agitation. Complete medium was prepared as explained above. After a 72 h growth period, animals were collected and then incubated with F-IVM at 10 µg/ml in complete liquid media seeded with OP50 at 2 1°C under gentle agitation. After 72 h, they were washed three times with M9 buffer to remove F-IVM and incubated for 4 hours in M9 buffer. Before imaging, samples were once again washed, and worms were paralyzed using Levamisole 2.5 mM and mounted on glass. F-IVM fluorescence was visualized and images were acquired with a Zeiss LSM 710 AxioObserver Plan-Apochromat 20x/0.8 M27 (Multi-Scale Imaging Facility, McGill University, Canada). F-IVM fluorescence was recorder as follows: diode laser 405 2%, emission wavelength 482 nm and detection wavelengths 414–550 nm with a detector gain of 620. Auto-fluorescence was recorded on worms that were not incubated with F-IVM. Images were acquired using ZEN 2.3 software. For each sample, the Mean Fluorescence Intensity, meaning the Integrated Intensity (A.U.) divided by the worm area (pixel²), was evaluated. It was then normalized with auto-fluorescence, i.e., the Mean Fluorescence Intensity of strains unlabeled with F-IVM. Experiments were replicated three times, and approximately 20–30 worms were analyzed per condition.

Statistical analysis

All experiments were conducted independently at least in triplicate, except for the F-IVM MA which was in duplicate. Results are expressed as mean ± standard deviation (S.D.) or ± standard error of the mean (S.E.M.). Statistical analyses were performed using the GraphPad Prism 8.4.2 software, and results were considered statistically significant when p < 0.05.

To evaluate the impact of each pgp deletion in the IVR10 background compared to the parental strain IVR10, a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple comparisons test was performed. Only key statistical results are presented in the study to maintain clarity and avoid excessive details. Development percentages at 4, 8, and 10 nM IVM of IVR10, IVR10(Δpgp-9) and IVR10(Δpgp-9)(ppgp-9::pgp-9) were pairwise compared with a two-way ANOVA followed by Tukey’s post-hoc multiple comparisons test. To assess the effect of pgp-9 invalidation in the N2B strain and of pgp-9 silencing in individual IVM-resistant strains, unpaired parametric t-tests were performed.

The expressions of Hco-pgp-9.1 in the three transgenic strains were individually compared to that of Cel-pgp-9 in IVR10 using unpaired parametric t-tests. LDAs with transgenic strains were statistically analyzed using a two-way ANOVA to assess the effects of the IVM concentrations and Hco-pgp-9.1 expression in IVR10(Δpgp-9) on larval development. Pairwise comparisons between IVR10(Δpgp-9) and IVR10(Δpgp-9) expressing the transgene at each concentration were performed using Sidak’s multiple comparisons test. The LDAs of each of the three transgenic strains were analyzed individually.

Statistical differences in mean pharyngeal pumping activity between N2B and IVR10 were assessed using an unpaired parametric t-test. F-IVM accumulation in N2B, IVR10 and IVR10(Δpgp-9) was statistically assessed with a one-way ANOVA followed by a Turkey’s post-hoc test.

Supporting information

S1 Fig. Expression of Cel-pgp-9 transgene.

Quantification of Cel-pgp-9 in IVR10(Δpgp-9) and IVR10 by single worm RT-qPCR. Data are expressed as fold change to the expression level of Cel-pgp-9 in the wild-type strain N2B. pgp-9 mRNA levels were normalized against the housekeeping gene tba-1 and are mean ± S.D. from three independent mRNA preparations per strain. One independent mRNA preparation corresponds to an RNA extraction from one single worm.

https://doi.org/10.1371/journal.ppat.1013355.s001

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S2 Fig. Impact of Cel-pgp-9 rescue in IVR10(Δpgp-9) on IVM tolerance.

Effect of transgene expression of Cel-pgp-9 on the tolerance of IVR10(Δpgp-9) to ivermectin (IVM) in a modified larval development assay (LDA), with IVR10 as a control. For each IVM concentration, the number of adult worms negative (i.e., IVR10(Δpgp-9)) and positive (i.e., Cel-pgp-9 transgene) for the extrachromosomal array was normalized to the number of adult worms of each population present in the DMSO well and expressed as a percentage. The percentages of animals reaching the adult stage for all concentrations were compared using pairwise using a two-way ANOVA followed by Tukey’s post-hoc multiple comparisons test). Data are mean ± S.D. from 3 independent experiments. Transgenic strain genotype: IVR10[Cel-pgp-9 -; pCel-pgp-9::Cel-pgp-9::3’UTR; pPD118.3].

https://doi.org/10.1371/journal.ppat.1013355.s002

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S3 Fig. pgp-9 silencing in IVM-resistant strains.

Effect of pgp-9 silencing on susceptibilities of IVM-resistant strains IVR10 (A), IVR10–2014 (B) and IVR10–2022 (C) to ivermectin (IVM) in a larval development assay (LDA). Worms were fed on bacteria carrying a plasmid producing double stranded RNA against pgp-9 plasmid (pgp-9 RNAi) or an empty plasmid as control (CTR RNAi). Values of dose-response curves represent the percentage of young adults maintaining motility within the presence of increasing doses of IVM. Data are mean ± S.D. from 3 independent experiments. IC50s for each strain are presented in S3 Table. The efficiency of pgp-9 knock-down was assessed by RT-qPCR (S4 Fig).

https://doi.org/10.1371/journal.ppat.1013355.s003

(TIF)

S4 Fig. pgp-9 mRNA expression following silencing.

Quantification of pgp-9 transcripts in IVR10, IVR10–2014, and IVR10–2022 following gene silencing. Real-time RT-qPCR analysis was applied after RNAi treatment with control RNAi or specific pgp-9 RNAi. pgp-9 mRNA levels in IVR10 strains are expressed as fold change relative to control RNAi. Data were normalized against tba-1 as an internal control and are mean ± S.D. from two independent RNA preparations for each strain.

https://doi.org/10.1371/journal.ppat.1013355.s004

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S5 Fig. Identity between Cel-PGP-9 and Hco-PGP-9.1.

Sequence alignments and percent identity matrices between Cel-PGP-9 (Accession Number: CE15714) and Hco-PGP-9.1 (Accession Number: HCON_00130050–00001) were conducted with the Clustal Omega tool. Symbols indicate: (*) conserved amino acid; (:) strong conservation following a substitution; (.) low similarity following a substitution; () no conservation. (A) Alignment was first performed with full predicted sequences and the percentage of identity between the two proteins was extracted. (B) Nucleotide binding domains (NBDs) were identified with the Scan Prosite tool and then manually curated from the sequences. Sequences were subjected a second time to Clustal Omega in order to highlight homology related to the substrate catalytic part of the protein (i.e., the transmembrane domains (TMDs).

https://doi.org/10.1371/journal.ppat.1013355.s005

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S6 Fig. Expression of Hco-pgp-9.1 transgene.

Quantification of Cel-pgp-9 in IVR10 and Hco-pgp-9.1 in the transgenic rescue strains, i.e., IVR10(Δpgp-9)-R#1, #2 and #3, by single worm RT-qPCR. Data are expressed as fold change to the expression level of Cel-pgp-9 in the wild-type strain N2B. pgp-9 mRNA levels were normalized against the housekeeping gene tba-1 and are mean ± S.D. from four independent mRNA preparations per strain. One independent mRNA preparation corresponds to an RNA extraction from one single worm. mRNA levels of Hco-pgp9.1 were compared to those of the IVR10 background strain as a reference (unpaired parametric t-test, ns).

https://doi.org/10.1371/journal.ppat.1013355.s006

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S7 Fig. Pharyngeal pumping activity of N2B and IVR10.

The pharyngeal pumping frequency (Hertz, Hz) corresponds to the average number of pharyngeal pumps recorded per worm for ~1 min. Data are presented as mean ± S.D. from three independent experiments with at least 10 worms per strain per replicate. No significant difference was observed between N2B and IVR10 (unpaired parametric t-test, p > 0.05).

https://doi.org/10.1371/journal.ppat.1013355.s007

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S8 Fig. Phylogenetic tree of Caenorhabditis elegans and Haemonchus contortus PGP protein sequences.

The PhyML phylogenetic tree was built using Seaview and iTOL (https://itol.embl.de/). Hsa-ABCB1 and Mmu-Pgp were used as an outgroup to root the tree. The scale bar represents the number of substitutions per site, and the node values correspond to branch support (approximate Likelihood Ratio Test). The corresponding multiple sequence alignment is presented in the S1 File.

https://doi.org/10.1371/journal.ppat.1013355.s008

(TIF)

S1 Table. Specific primers for Caenorhabditis elegans or Haemonchus contortus genes targeted by RT-qPCR.

https://doi.org/10.1371/journal.ppat.1013355.s009

(DOCX)

S2 Table. Primers used for cloning and DNA amplification in this study. a Primers used for subcloning into pMini 2.0; b primers used for cloning into pPD95.75; c primers used for Cel-pgp-9 amplification, including the promoter and 3’UTR of the gene.

In bold: restriction sites. Underlined: overhangs for Gibson cloning.

https://doi.org/10.1371/journal.ppat.1013355.s010

(DOCX)

S3 Table. Susceptibilities to ivermectin (IVM) of IVM selected strains IVR10, IVR10–2014, and IVR10–2022 following pgp-9 silencing on larval development assay (LDA).

IC50: inhibitory concentration 50%. a p < 0.001, b p < 0.05 strain fed on pgp-9 RNAi versus strain fed on control RNAi (unpaired parametric t-test).

https://doi.org/10.1371/journal.ppat.1013355.s011

(DOCX)

S4 Table. Comparison of molecular parameters between IVM and F-IVM, as modeled using ChemDraw Professional 17.1 and Chem3D 17.1 (PerkinElmer Informatics).

Calculation method: a ChemPropStd; b CLogP Driver; c Molecular Networks; d Molecular Topology; e MM2 minimization.

https://doi.org/10.1371/journal.ppat.1013355.s012

(DOCX)

S5 Table. Evidence of Cel-pgp-9/PGP-9 localization from the literature.

https://doi.org/10.1371/journal.ppat.1013355.s013

(DOCX)

S1 File. Sequence alignment of Cel and Hco-PGP sequences.

Multiple sequence alignment was performed with the Clustal Omega method using Seaview and served as the basis for the phylogenetic tree. Cel-pgp-15 is a pseudogene and was therefore not included.

https://doi.org/10.1371/journal.ppat.1013355.s014

(PDF)

Acknowledgments

We thank Rémy Betous for his support and advice in Molecular Biology, and Lucas Barat for the numerous scientific discussions and his punctual assistance. We would like to thank the McGill Institute of Parasitology Institute and the Multi-Scale Imaging Facility where some of the transgenic strains and part of the confocal images were respectively performed. We also thank Hua Che for the technical support at McGill and Cody Hart, who facilitated the acquisition of microinjection skills. The rest of the transgenic strains were generated at SEGiCel (SFR Santé Lyon Est CNRS UAR 3453, Lyon, France) with the support of CNRS and IBiSA. We thank Thomas Boulin, Margaux Gibert, and Marielle Limoges for their support. Some of the confocal imaging experiments were also performed at the Infinity-INSERM UMR1291 core facility connected to the Toulouse Réseau Imagerie network, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04). For that, we thank Simon Lachambre for his assistance with the confocal imaging systems. We also thank Cécile Pouzet for the technical assistance at the TRI-FRAIB Imaging Facility (Université de Toulouse-CNRS).

References

  1. 1. Specht S, Keiser J. Helminth infections: Enabling the World Health Organization Road Map. Int J Parasitol. 2023;53(8):411–4. pmid:36549443
  2. 2. Anisuzzaman, Tsuji N. Schistosomiasis and hookworm infection in humans: Disease burden, pathobiology and anthelmintic vaccines. Parasitol Int. 2020;75:102051. pmid:31911156
  3. 3. Zajac AM, Garza J. Biology, epidemiology, and control of gastrointestinal nematodes of small ruminants. Vet Clin North Am Food Anim Pract. 2020;36:73–87.
  4. 4. Charlier J, Rinaldi L, Musella V, Ploeger HW, Chartier C, Vineer HR, et al. Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe. Prev Vet Med. 2020;182:105103. pmid:32750638
  5. 5. Martin RJ, Robertson AP, Choudhary S. Ivermectin: An Anthelmintic, an Insecticide, and Much More. Trends Parasitol. 2021;37(1):48–64. pmid:33189582
  6. 6. Kotze AC, Prichard RK. Anthelmintic Resistance in Haemonchus contortus. Advances in Parasitology. Elsevier. 2016:397–428.
  7. 7. Lustigman S, McCarter JP. Ivermectin resistance in Onchocerca volvulus: toward a genetic basis. PLoS Negl Trop Dis. 2007;1(1):e76. pmid:17989789
  8. 8. Charlier J, Thamsborg SM, Bartley DJ, Skuce PJ, Kenyon F, Geurden T, et al. Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs. Transbound Emerg Dis. 2018;65 Suppl 1:217–34. pmid:29124904
  9. 9. Lespine A, Blancfuney C, Prichard R, Alberich M. P-glycoproteins in anthelmintic safety, efficacy, and resistance. Trends Parasitol. 2025;41(1):334.
  10. 10. Raza A, Kopp SR, Bagnall NH, Jabbar A, Kotze AC. Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae. International Journal for Parasitology: Drugs and Drug Resistance. 2016;6: 103–15.
  11. 11. Pacheco PA, Louvandini H, Giglioti R, Wedy BCR, Ribeiro JC, Verissimo CJ, et al. Phytochemical modulation of P-Glycoprotein and its gene expression in an ivermectin-resistant Haemonchus contortus isolate in vitro. Vet Parasitol. 2022;305:109713. pmid:35597694
  12. 12. Tuersong W, Zhou C, Wu S, Qin P, Wang C, Di W, et al. Comparative analysis on transcriptomics of ivermectin resistant and susceptible strains of Haemonchus contortus. Parasit Vectors. 2022;15(1):159. pmid:35524281
  13. 13. Mate L, Ballent M, Cantón C, Lanusse C, Ceballos L, Alvarez LLI, et al. ABC-transporter gene expression in ivermectin-susceptible and resistant Haemonchus contortus isolates. Veterinary Parasitology. 2022;302:109647.
  14. 14. Gerhard AP, Krücken J, Heitlinger E, Janssen IJI, Basiaga M, Kornaś S, et al. The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens. Sci Rep. 2020;10(1):13586. pmid:32788636
  15. 15. Choi Y-J, Bisset SA, Doyle SR, Hallsworth-Pepin K, Martin J, Grant WN, et al. Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta. PLoS Genet. 2017;13(6):e1006857. pmid:28644839
  16. 16. Mealey KL. Therapeutic implications of the MDR-1 gene. J Vet Pharmacol Ther. 2004;27(5):257–64. pmid:15500562
  17. 17. David M, Lebrun C, Duguet T, Talmont F, Beech R, Orlowski S, et al. Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13. Int J Parasitol Drugs Drug Resist. 2018;8(1):145–57. pmid:29571165
  18. 18. Godoy P, Che H, Beech RN, Prichard RK. Characterisation of P-glycoprotein-9.1 in Haemonchus contortus. Parasit Vectors. 2016;9:52. pmid:26822677
  19. 19. Mani T, Bourguinat C, Keller K, Ashraf S, Blagburn B, Prichard RK. Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein. Int J Parasitol. 2016;46(10):631–40. pmid:27172882
  20. 20. Jesudoss Chelladurai JRJ, Jones DE, Brewer MT. Characterization of a P-glycoprotein drug transporter from Toxocara canis with a novel pharmacological profile. Int J Parasitol Drugs Drug Resist. 2021;17:191–203. pmid:34753107
  21. 21. Gerhard AP, Krücken J, Neveu C, Charvet CL, Harmache A, von Samson-Himmelstjerna G. Pharyngeal Pumping and Tissue-Specific Transgenic P-Glycoprotein Expression Influence Macrocyclic Lactone Susceptibility in Caenorhabditis elegans. Pharmaceuticals. 2021;14:153.
  22. 22. Janssen IJI, Krücken J, Demeler J, von Samson-Himmelstjerna G. Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans. Int J Parasitol Drugs Drug Resist. 2015;5(2):44–7. pmid:25905032
  23. 23. Raza A, Kopp SR, Jabbar A, Kotze AC. Effects of third generation P-glycoprotein inhibitors on the sensitivity of drug-resistant and -susceptible isolates of Haemonchus contortus to anthelmintics in vitro. Vet Parasitol. 2015;211(1–2):80–8. pmid:25986327
  24. 24. Jesudoss Chelladurai JRJ, Martin KA, Vardaxis P, Reinemeyer C, Vijayapalani P, Robertson AP, et al. Repertoire of P-glycoprotein drug transporters in the zoonotic nematode Toxocara canis. Sci Rep. 2023;13(1):4971. pmid:36973306
  25. 25. James CE, Davey MW. Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans. Int J Parasitol. 2009;39(2):213–20. pmid:18708066
  26. 26. Janssen IJI, Krücken J, Demeler J, von Samson-Himmelstjerna G. Caenorhabditis elegans: modest increase of susceptibility to ivermectin in individual P-glycoprotein loss-of-function strains. Exp Parasitol. 2013;134(2):171–7. pmid:23518455
  27. 27. Ménez C, Alberich M, Kansoh D, Blanchard A, Lespine A. Acquired Tolerance to Ivermectin and Moxidectin after Drug Selection Pressure in the Nematode Caenorhabditis elegans. Antimicrob Agents Chemother. 2016;60(8):4809–19. pmid:27246778
  28. 28. Dube F, Hinas A, Delhomme N, Åbrink M, Svärd S, Tydén E. Transcriptomics of ivermectin response in Caenorhabditis elegans: Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain. PLoS One. 2023;18(5):e0285262. pmid:37141255
  29. 29. Freeman AS, Nghiem C, Li J, Ashton FT, Guerrero J, Shoop WL, et al. Amphidial structure of ivermectin-resistant and susceptible laboratory and field strains of Haemonchus contortus. Vet Parasitol. 2003;110(3–4):217–26. pmid:12482650
  30. 30. Urdaneta-Marquez L, Bae SH, Janukavicius P, Beech R, Dent J, Prichard R. A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus. Int J Parasitol. 2014;44(14):1063–71. pmid:25224687
  31. 31. Ménez C, Alberich M, Courtot E, Guegnard F, Blanchard A, Aguilaniu H, et al. The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes. PLoS Pathog. 2019;15(2):e1007598. pmid:30759156
  32. 32. Guerrero GA, Derisbourg MJ, Mayr FA, Wester LE, Giorda M, Dinort JE, et al. NHR-8 and P-glycoproteins uncouple xenobiotic resistance from longevity in chemosensory C. elegans mutants. Elife. 2021;10:e53174. pmid:34448454
  33. 33. Dent JA, Smith MM, Vassilatis DK, Avery L. The genetics of ivermectin resistance in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2000;97(6):2674–9. pmid:10716995
  34. 34. Alberich M, Garcia M, Petermann J, Blancfuney C, Jouffroy S, Jacquiet P, et al. Evaluation of nematode susceptibility and resistance to anthelmintic drugs with a WMicrotracker motility assay. Sci Rep. 2025;15(1):17968. pmid:40410413
  35. 35. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 2000;408(6810):325–30. pmid:11099033
  36. 36. Kamal M, Tokmakjian L, Knox J, Han D, Moshiri H, Magomedova L, et al. PGP-14 establishes a polar lipid permeability barrier within the C. elegans pharyngeal cuticle. PLoS Genet. 2023;19(11):e1011008. pmid:37930961
  37. 37. Ghaddar A, Armingol E, Huynh C, Gevirtzman L, Lewis NE, Waterston R, et al. Whole-body gene expression atlas of an adult metazoan. Sci Adv. 2023;9(25):eadg0506. pmid:37352352
  38. 38. Zhao Z, Sheps JA, Ling V, Fang LL, Baillie DL. Expression analysis of ABC transporters reveals differential functions of tandemly duplicated genes in Caenorhabditis elegans. J Mol Biol. 2004;344(2):409–17. pmid:15522294
  39. 39. Hartman JH, Widmayer SJ, Bergemann CM, King DE, Morton KS, Romersi RF, et al. Xenobiotic metabolism and transport in Caenorhabditis elegans. J Toxicol Environ Health B Crit Rev. 2021;24(2):51–94. pmid:33616007
  40. 40. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. Cell. 2021;184(16):4329–4347.e23. pmid:34237253
  41. 41. Cao J, Packer JS, Ramani V, Cusanovich DA, Huynh C, Daza R, et al. Comprehensive single-cell transcriptional profiling of a multicellular organism. Science. 2017;357(6352):661–7. pmid:28818938
  42. 42. Gerhard AP. The role of nematode P-glycoproteins in the mechanisms of macrocyclic lactone resistance. Freien Universität Berlin. 2021. https://refubium.fu-berlin.de/handle/fub188/31742
    • 43. Wolstenholme AJ. Glutamate-gated chloride channels. J Biol Chem. 2012;287(48):40232–8. pmid:23038250
    • 44. AlGusbi S, Krücken J, Ramünke S, von Samson-Himmelstjerna G, Demeler J. Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes. Int J Parasitol. 2014;44(9):647–58. pmid:24907555
    • 45. Doyle SR, Laing R, Bartley D, Morrison A, Holroyd N, Maitland K, et al. Genomic landscape of drug response reveals mediators of anthelmintic resistance. Cell Rep. 2022;41(3):111522. pmid:36261007
    • 46. Laing R, Doyle SR, McIntyre J, Maitland K, Morrison A, Bartley DJ, et al. Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode. PLoS Pathog. 2022;18(6):e1010545. pmid:35696434
    • 47. Doyle SR, Tracey A, Laing R, Holroyd N, Bartley D, Bazant W, et al. Genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm. Commun Biol. 2020;3(1):656. pmid:33168940
    • 48. Evans TC. Transformation and microinjection. WormBook: The Online Review of C elegans Biology. https://www.ncbi.nlm.nih.gov/books/NBK19648/. 2006.
    • 49. Ly K, Reid SJ, Snell RG. Rapid RNA analysis of individual Caenorhabditis elegans. MethodsX. 2015;2:59–63. pmid:26150972